CD248 induces a maladaptive unfolded protein response in diabetic kidney disease
Authors: Shruthi Krishnan, Jayakumar Manoharan, Hongjie Wang, Dheerendra Gupta, Sameen Fatima, Yanfei Yu, Akash Mathew, Zhen Li, Shrey Kohli, Constantin Schwab, Antje Körner, Peter R. Mertens, Peter Nawroth, Khurrum Shahzad, Michael Naumann, Berend Isermann, Ronald Biemann
Journal: Kidney International (2022)

Dysfunction of mesangial cells plays a major role in the pathogenesis of diabetic kidney disease (DKD), the leading cause of kidney failure. However, the underlying molecular mechanisms are incompletely understood. By unbiased gene expression analysis of glucose-exposed mesangial cells, we identified the transmembrane receptor CD248 as the most upregulated gene, and the maladaptive unfolded protein response (UPR) as one of the most stimulated pathways. Upregulation of CD248 was further confirmed in glucose-stressed mesangial cells in vitro, in kidney glomeruli isolated from diabetic mice (streptozotocin; STZ and db/db models, representing type 1 and type 2 diabetes mellitus, respectively) in vivo, and in glomerular kidney sections from patients with DKD. Time course analysis revealed that glomerular CD248 induction precedes the onset of albuminuria, mesangial matrix expansion and maladaptive UPR activation (hallmarked by transcription factor C/EBP homologous protein (CHOP) induction) but is paralleled by loss of the adaptive UPR regulator spliced X box binding protein (XBP1). Mechanistically, CD248 promoted maladaptive UPR signaling via inhibition of the inositol requiring enzyme 1α (IRE1α)-mediated transcription factor XBP1 splicing in vivo and in vitro. CD248 induced a multiprotein complex comprising heat shock protein 90, BH3 interacting domain death agonist (BID) and IRE1α, in which BID impedes IRE1α-mediated XBP1 splicing and induced CHOP mediated maladaptive UPR signaling. While CD248 knockout ameliorated DKD-associated glomerular dysfunction and reverses maladaptive unfolded protein response signaling, concomitant XBP1 deficiency abolished the protective effect in diabetic CD248 knockout mice, supporting a functional interaction of CD248 and XBP1 in vivo. Hence, CD248 is a novel mesangial cell receptor inducing maladaptive UPR signaling in DKD. Dysfunction of mesangial cells plays a major role in the pathogenesis of diabetic kidney disease (DKD), the leading cause of kidney failure. However, the underlying molecular mechanisms are incompletely understood. By unbiased gene expression analysis of glucose-exposed mesangial cells, we identified the transmembrane receptor CD248 as the most upregulated gene, and the maladaptive unfolded protein response (UPR) as one of the most stimulated pathways. Upregulation of CD248 was further confirmed in glucose-stressed mesangial cells in vitro, in kidney glomeruli isolated from diabetic mice (streptozotocin; STZ and db/db models, representing type 1 and type 2 diabetes mellitus, respectively) in vivo, and in glomerular kidney sections from patients with DKD. Time course analysis revealed that glomerular CD248 induction precedes the onset of albuminuria, mesangial matrix expansion and maladaptive UPR activation (hallmarked by transcription factor C/EBP homologous protein (CHOP) induction) but is paralleled by loss of the adaptive UPR regulator spliced X box binding protein (XBP1). Mechanistically, CD248 promoted maladaptive UPR signaling via inhibition of the inositol requiring enzyme 1α (IRE1α)-mediated transcription factor XBP1 splicing in vivo and in vitro. CD248 induced a multiprotein complex comprising heat shock protein 90, BH3 interacting domain death agonist (BID) and IRE1α, in which BID impedes IRE1α-mediated XBP1 splicing and induced CHOP mediated maladaptive UPR signaling. While CD248 knockout ameliorated DKD-associated glomerular dysfunction and reverses maladaptive unfolded protein response signaling, concomitant XBP1 deficiency abolished the protective effect in diabetic CD248 knockout mice, supporting a functional interaction of CD248 and XBP1 in vivo. Hence, CD248 is a novel mesangial cell receptor inducing maladaptive UPR signaling in DKD. Translational StatementWe identified the type I transmembrane receptor CD248 as an inducer of maladaptive unfolded protein response (UPR) in mesangial cells. Diabetic kidney disease (DKD)–associated induction of CD248 inhibits cytoprotective IRE1α-mediated XBP1 splicing, whereas CD248 knockout ameliorates DKD-associated glomerular dysfunction and reverses maladaptive UPR signaling. The enhanced CD248 expression in DKD, in combination with the lack of a detrimental phenotype in CD248-deficient mice, suggests that CD248 is an attractive therapeutic target. Antibody-based therapeutic approaches targeting CD248 (ontuxizumab, scFV-78Fc) have been developed, have been evaluated in cancer patients, and are well tolerated. Repurposing these antibodies for DKD may be an attractive therapeutic option for patients with DKD. We identified the type I transmembrane receptor CD248 as an inducer of maladaptive unfolded protein response (UPR) in mesangial cells. Diabetic kidney disease (DKD)–associated induction of CD248 inhibits cytoprotective IRE1α-mediated XBP1 splicing, whereas CD248 knockout ameliorates DKD-associated glomerular dysfunction and reverses maladaptive UPR signaling. The enhanced CD248 expression in DKD, in combination with the lack of a detrimental phenotype in CD248-deficient mice, suggests that CD248 is an attractive therapeutic target. Antibody-based therapeutic approaches targeting CD248 (ontuxizumab, scFV-78Fc) have been developed, have been evaluated in cancer patients, and are well tolerated. Repurposing these antibodies for DKD may be an attractive therapeutic option for patients with DKD. Diabetic kidney disease (DKD) is a leading diabetic complication, frequently causing kidney failure, and it is the strongest predictor of mortality in diabetic patients.1Fouli G.E. Gnudi L. The future: experimental therapies for renal disease in diabetes.Nephron. 2019; 143: 3-7Crossref PubMed Scopus (16) Google Scholar Early stages of DKD are, in principle, reversible,2Gaede P. Tarnow L. Vedel P. et al.Remission to normoalbuminuria during multifactorial treatment preserves kidney function in patients with type 2 diabetes and microalbuminuria.Nephrol Dial Transplant. 2004; 19: 2784-2788Crossref PubMed Scopus (202) Google Scholar yet specific medical strategies exploiting this therapeutic window are scarce.3Reidy K. Kang H.M. Hostetter T. Susztak K. Molecular mechanisms of diabetic kidney disease.J Clin Invest. 2014; 124: 2333-2340Crossref PubMed Scopus (609) Google Scholar An emerging molecular mechanism linked with DKD is the maladaptive unfolded protein response (UPR). The endoplasmic reticulum (ER) conveys important functions in maintaining protein and calcium homeostasis. DKD is frequently associated with ER dysfunction leading to activation of the UPR, and recent preclinical studies suggest that maladaptive UPR signaling plays a pivotal role in the pathogenesis of DKD.4Gnudi L. Coward R.J.M. Long D.A. Diabetic nephropathy: perspective on novel molecular mechanisms.Trends Endocrinol Metab. 2016; 27: 820-830Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 5Madhusudhan T. Wang H.G. Dong W. et al.Defective podocyte insulin signalling through p85-XBP1 promotes ATF6-dependent maladaptive ER-stress response in diabetic nephropathy.Nat Commun. 2015; 6: 6496Crossref PubMed Scopus (129) Google Scholar, 6Shahzad K. Fatima S. Al-Dabet M.M. et al.CHOP-ASO ameliorates glomerular and tubular damage on top of ACE inhibition in diabetic kidney disease.J Am Soc Nephrol. 2021; 32: 3066-3079Crossref Scopus (13) Google Scholar Strategies restoring ER homeostasis or interfering with the maladaptive UPR have been proposed as attractive therapeutic approaches in DKD.5Madhusudhan T. Wang H.G. Dong W. et al.Defective podocyte insulin signalling through p85-XBP1 promotes ATF6-dependent maladaptive ER-stress response in diabetic nephropathy.Nat Commun. 2015; 6: 6496Crossref PubMed Scopus (129) Google Scholar, 6Shahzad K. Fatima S. Al-Dabet M.M. et al.CHOP-ASO ameliorates glomerular and tubular damage on top of ACE inhibition in diabetic kidney disease.J Am Soc Nephrol. 2021; 32: 3066-3079Crossref Scopus (13) Google Scholar, 7Cybulsky A.V. Endoplasmic reticulum stress, the unfolded protein response and autophagy in kidney diseases.Nat Rev Nephrol. 2017; 13: 681-696Crossref PubMed Scopus (342) Google Scholar, 8Marquardt A. Al-Dabet M.M. Ghosh S. et al.Farnesoid X receptor agonism protects against diabetic tubulopathy: potential add-on therapy for diabetic nephropathy.J Am Soc Nephrol. 2017; 28: 3182-3189Crossref PubMed Scopus (51) Google Scholar Toxin-mediated impairment of ER protein folding simultaneously activates the 3 major pathways initiated by ER membrane-resident proteins: inositol-requiring enzyme 1 (IRE1α), double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK), and activating transcription factor 6 (ATF6).9Ron D. Walter P. Signal integration in the endoplasmic reticulum unfolded protein response.Nat Rev Mol Cell Biol. 2007; 8: 519-529Crossref PubMed Scopus (5134) Google Scholar,10Rutkowski D.T. Arnold S.M. Miller C.N. et al.Adaptation to ER stress is mediated by differential stabilities of pro-survival and pro-apoptotic mRNAs and proteins.PLoS Biol. 2006; 4: e374Crossref PubMed Scopus (654) Google Scholar However, under disease conditions, these pathways are differentially regulated, exerting either protective or detrimental effects on diabetes mellitus-associated complications.5Madhusudhan T. Wang H.G. Dong W. et al.Defective podocyte insulin signalling through p85-XBP1 promotes ATF6-dependent maladaptive ER-stress response in diabetic nephropathy.Nat Commun. 2015; 6: 6496Crossref PubMed Scopus (129) Google Scholar,11Back S.H. Kaufman R.J. Endoplasmic reticulum stress and type 2 diabetes.Annu Rev Biochem. 2012; 81: 767-793Crossref PubMed Scopus (449) Google Scholar The most highly conserved UPR branch, the IRE1α/X box binding protein 1 (XBP1) branch, has context-dependent effects. This pathway maintains cellular homeostasis and performs cytoprotective functions in most disease models.12Kaser A. Lee A.-H. Franke A. et al.XBP1 links ER stress to intestinal inflammation and confers genetic risk for human inflammatory bowel disease.Cell. 2008; 134: 743-756Abstract Full Text Full Text PDF PubMed Scopus (1133) Google Scholar,13Akiyama M. Liew C.W. Lu S. et al.X-box binding protein 1 is essential for insulin regulation of pancreatic α-cell function.Diabetes. 2013; 62: 2439-2449Crossref PubMed Scopus (47) Google Scholar Upon sensing unfolded proteins, the ER transmembrane sensor IRE1α oligomerizes, autophosphorylates, and utilizes its endoribonuclease activity to excise an intron from the mRNA of the transcription factor XBP1, generating spliced XBP1 (sXBP1).14Calfon M. Zeng H. Urano F. et al.IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA.Nature. 2002; 415: 92-96Crossref PubMed Scopus (2237) Google Scholar sXBP1 translocates into the nucleus and binds to target sequences in the regulatory regions of ER-associated degradation and ER chaperone genes.15Lee A.-H. Iwakoshi N.N. Glimcher L.H. XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response.Mol Cell Biol. 2003; 23: 7448-7459Crossref PubMed Scopus (1700) Google Scholar,16Madhusudhan T. Wang H. Ghosh S. et al.Signal integration at the PI3K-p85-XBP1 hub endows coagulation protease activated protein C with insulin-like function.Blood. 2017; 130: 1445-1455Crossref PubMed Scopus (25) Google Scholar IRE1α activity is regulated by the assembly of dynamic multiprotein complexes named UPRosomes, which are cell- and context-specific.17Urra H. Pihán P. Hetz C. The UPRosome—decoding novel biological outputs of IRE1α function.J Cell Sci. 2020; 133: jcs218107Crossref PubMed Scopus (29) Google Scholar IRE1α/XBP1 maintains cellular homeostasis and performs cytoprotective functions in most disease models,12Kaser A. Lee A.-H. Franke A. et al.XBP1 links ER stress to intestinal inflammation and confers genetic risk for human inflammatory bowel disease.Cell. 2008; 134: 743-756Abstract Full Text Full Text PDF PubMed Scopus (1133) Google Scholar,13Akiyama M. Liew C.W. Lu S. et al.X-box binding protein 1 is essential for insulin regulation of pancreatic α-cell function.Diabetes. 2013; 62: 2439-2449Crossref PubMed Scopus (47) Google Scholar and impaired XBP1 signaling provokes a maladaptive ER response characterized by enhanced ATF6 and C/EBP homologous protein (CHOP) signaling.5Madhusudhan T. Wang H.G. Dong W. et al.Defective podocyte insulin signalling through p85-XBP1 promotes ATF6-dependent maladaptive ER-stress response in diabetic nephropathy.Nat Commun. 2015; 6: 6496Crossref PubMed Scopus (129) Google Scholar In podocytes, insulin signaling regulates UPR-induced XBP1 splicing, and disruption of insulin signaling is causatively linked to the maladaptive UPR in podocytes.5Madhusudhan T. Wang H.G. Dong W. et al.Defective podocyte insulin signalling through p85-XBP1 promotes ATF6-dependent maladaptive ER-stress response in diabetic nephropathy.Nat Commun. 2015; 6: 6496Crossref PubMed Scopus (129) Google Scholar Podocyte-specific IRE1 knockout accelerates proteinuria, glomerular fibrosis, and podocyte cell death in diabetic mice.18Xie L. Guo K. Lu S. et al.Diabetic nephropathy in mice is aggravated by the absence of podocyte IRE1 and is correlated with reduced kidney ADH1 expression.Ann Transl Med. 2021; 9: 636Crossref PubMed Google Scholar Additionally, a high glucose level reduces sXBP1 expression in renal mesangial cells in vitro, which increases reactive oxygen species (ROS) production and expression of extracellular matrix proteins.19Shao D. Liu J. Ni J. et al.Suppression of XBP1S mediates high glucose-induced oxidative stress and extracellular matrix synthesis in renal mesangial cell and kidney of diabetic rats.PloS One. 2013; 8e56124Crossref Scopus (31) Google Scholar Furthermore, maladaptive ER stress signaling in DKD is causally linked to increased glomerular and tubular expression of the proinflammatory transcription factor CHOP,6Shahzad K. Fatima S. Al-Dabet M.M. et al.CHOP-ASO ameliorates glomerular and tubular damage on top of ACE inhibition in diabetic kidney disease.J Am Soc Nephrol. 2021; 32: 3066-3079Crossref Scopus (13) Google Scholar which promotes kidney fibrosis. Identification of specific receptor-mediated molecular pathways underlying DKD-associated glomerular cell dysfunction may lead to new therapeutic approaches for DKD. For example, loss of the transmembrane receptor thrombomodulin impairs generation of activated protein C. Activated protein C is a cytoprotective protease, mimicking insulin-dependent suppression of UPR in DKD, and thus conveying nephroprotective effects in DKD.16Madhusudhan T. Wang H. Ghosh S. et al.Signal integration at the PI3K-p85-XBP1 hub endows coagulation protease activated protein C with insulin-like function.Blood. 2017; 130: 1445-1455Crossref PubMed Scopus (25) Google Scholar Although thrombomodulin-dependent activated protein C generation is protective against DKD, the transmembrane receptors that sense the "diabetic milieu" and induce a maladaptive UPR response remain unknown. A molecule structurally related to thrombomodulin is CD248 (also called endosialin), a type I transmembrane glycoprotein.20Christian S. Ahorn H. Koehler A. et al.Molecular cloning and characterization of endosialin, a C-type lectin-like cell surface receptor of tumor endothelium.J Biol Chem. 2001; 276: 7408-7414Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar CD248 is expressed by cells of mesenchymal origin, such as mesangial cells, stromal cells, fibroblasts, vascular smooth muscle cells, and pericytes, but not by endothelial cells.21Rettig W.J. Garin-Chesa P. Healey J.H. et al.Identification of endosialin, a cell surface glycoprotein of vascular endothelial cells in human cancer.Proc Natl Acad Sci U S A. 1992; 89: 10832-10836Crossref PubMed Scopus (218) Google Scholar,22MacFadyen J.R. Haworth O. Robertston D. et al.Endosialin (TEM1, CD248) is a marker of stromal fibroblasts and is not selectively expressed on tumour endothelium.FEBS Lett. 2005; 579: 2569-2575Crossref PubMed Scopus (146) Google Scholar Postnatally, CD248 mRNA expression decreases in most organs but persists in the kidney, specifically in glomerular mesangial cells and vascular pericytes.23Huang H.-P. Hong C.-L. Kao C.-Y. et al.Gene targeting and expression analysis of mouse Tem1/endosialin using a lacZ reporter.Gene Expr Patterns. 2011; 11: 316-326Crossref PubMed Scopus (25) Google Scholar CD248 knockout mice are protected from renal fibrosis in unilateral ureteral obstruction and unilateral ischemia–reperfusion injury models.24Pai C.-H. Lin S.-R. Liu C.-H. et al.Targeting fibroblast CD248 attenuates CCL17-expressing macrophages and tissue fibrosis.Sci Rep. 2020; 10: 16772Crossref PubMed Scopus (17) Google Scholar Single-cell transcriptomic analysis indicated that CD248 is upregulated in mesangial cells in human DKD, but not in other renal cells.25Wilson P.C. Wu H. Kirita Y. et al.The single-cell transcriptomic landscape of early human diabetic nephropathy.Proc Natl Acad Sci U S A. 2019; 116: 19619-19625Crossref PubMed Scopus (266) Google Scholar Whether CD248 induces renal changes in DKD, and whether this relates to maladaptive UPR in DKD, remains unknown. To identify possible mediators of maladaptive UPR in mesangial cells in DKD, we conducted hypothesis-free gene expression analyses in control and glucose-challenged mesangial cells. See the Supplementary Methods for detailed information. C57BL/6J (wild-type) mice (obtained from The Jackson Laboratory), nondiabetic C57BLKsJ-db/+ (db/m) and diabetic C57BL/KsJ-db/db (db/db) mice (both obtained from Janvier), CD248–/– mice (provided by David. L. Huso), and XBP1+/– mice (provided by L. Glimcher) were used for this study. CD248–/– and XBP1+/– mice were crossed to generate the CD248–/– x XBP1+/– line. All mice were backcrossed onto the C57BL/6J background for at least 9 generations and were routinely maintained on the C57BL/6J background. Only the CD248–/– and CD248–/– x XBP1+/– littermates were used as nondiabetic control mice in the current study. Mice were randomly assigned to the experimental groups. Knockout alleles of the CD248 and Xbp1 genes were routinely verified by polymerase chain reaction analyses of tail DNA or ear biopsies. Animal experiments were conducted following the standards and procedures approved by the local animal care and use committee (Landesverwaltungsamt Halle and Landesverwaltungsamt Leipzig, Germany). Persistent hyperglycemia representing type 1 diabetes mellitus was induced in 8-week-old male mice by intraperitoneal administration of streptozotocin (STZ; 60 mg/kg, freshly dissolved in 0.05 M sterile sodium citrate [pH 4.5]), on 5 successive days. Only mice with persistently elevated glucose levels 2 weeks post–STZ injection were used as diabetic mice. Mice that did not develop hyperglycemia despite STZ injection (dropout rate about 15%) were included in the control group and did not differ from control mice that did not receive STZ. Mice were considered diabetic if their blood glucose levels were above 300 mg/dl (16.7 mM), at 16–25 days after the last STZ injection. Blood glucose levels of mice were measured under random conditions. Age-matched littermates served as controls. Immortalized mouse mesangial cells (MES13) and human renal mesangial cells were obtained from ATCC and ScienCell, respectively. MES13 cells were cultured using a 3:1 mixture of ATCC-formulated Dulbecco's modified Eagle's medium and Ham's F12 medium supplemented with 14 mM HEPES, 5% fetal bovine serum, and 10 U/ml penicillin/streptomycin. Human renal mesangial cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 10 U/ml penicillin/streptomycin and insulin-transferrin-selenium (ITS; Sigma). Conditionally immortalized human glomerular endothelial cells (h.GECs), and human and mouse podocytes (h.PODs and PODs), were routinely grown on cell culture–grade plates at 33 °C in the presence of interferon γ (10 U/ml) to enhance expression of a thermosensitive T antigen. These conditions are optimum for cells to proliferate and remain undifferentiated. To induce differentiation, thermoswitching was done at 37 °C in the absence of interferon γ (for h.PODs and PODs) and with the addition of 100 ng/ml vascular endothelial growth factor (for h.GECs). Experiments were performed after 7 days of differentiation for h.GECs, and after14 days of differentiation for h.PODs and PODs. Mouse glomerular endothelial cells (GECs, Cell Biologics) were cultured at 37 °C in a humidified 5% CO2 incubator in M1168 mouse endothelial cell medium, with growth factors as provided by the manufacturer. BUMPT cells were cultured according to an established protocol.26Smith S.W. Eardley K.S. Croft A.P. et al.CD248+ stromal cells are associated with progressive chronic kidney disease.Kid Int. 2011; 80: 199-207Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar GECs and h.GECs were cultured on 0.02% gelatin coated plates at 37 °C in M-6115 and H-6115 media (from Cell biologicals). The cell lines were regularly tested and found to be negative for mycoplasma contamination. MES13 cells were transiently transfected with short hairpin RNA against CD248, small, interfering RNA against HSP90, CD248–C-terminal FLAG-tagged plasmid, and BH3 interacting domain death agonist (BID)-C-terminal HA-tagged plasmid using Turbofect (Thermo Scientific). The different renal cell lines were exposed to high glucose (HG, 25 mM), mannitol osmolarity control (M, 25 mM), or normal glucose (NG, 5 mM) for 48 hours and then harvested for RNA isolation. Subsets of cells were exposed to 4μ8C (2μM, IRE1α RNAse inhibitor, IRE1αi) for 5 hours. The data are summarized as mean and SEM values. Either the Kolmogorov–Smirnov test or the D'Agostino–Pearson normality test was used to determine whether the data followed a Gaussian distribution. Statistical analyses were performed using the Student's t test or analysis of variance, as indicated in the figure legends. Analysis of variance comparisons were subjected to post hoc correction with Tukey's multiple-comparison test. Prism 5 software (www.graphpad.com) was used for statistical analyses. Statistical significance was accepted at P < 0.05. We performed transcriptomic analysis of a murine mesangial cell line (MES13) exposed to normal-glucose or high-glucose conditions by RNA sequencing. A total of 2943 genes were differentially regulated under high-glucose conditions, with 614 genes upregulated and 2329 genes downregulated (Figure 1a ). Differential gene expression analysis identified the type 1 transmembrane receptor CD248 as the most upregulated gene in high glucose–stressed mesangial cells (Figure 1a). Gene ontology–based molecular pathway analysis of the upregulated genes revealed that protein translation (P value of 3.6E-6), response to ER stress (P value of 1.6E-6), and UPR (P value of 1.4E-6) were the most highly upregulated pathways (as shown in red bars, Figure 1b). Several genes that code for ER stress sensing, UPR, ER-associated degradation, or ER chaperone proteins, such as Ddit3, Xbp1, Atf6, Herpud1, Trib3, Dnajb9, and Hspa5, were found to be among the most upregulated targets (Supplementary Table S1). Selected results from the expression analyses were verified by quantitative polymerase chain reaction analysis, confirming the induction of CD248, Ddit3, Atf6, Herpud1, Trib3, and Hspa5, and suppression of Pmepa1, Id1, and Aqp1 (Figure 1c). To validate whether CD248 protein expression is induced by high glucose conditions in mesangial cells, we used human renal mesangial cells, murine mesangial MES13 cells, and isolated primary murine mesangial cells (Figure 1d–f; Supplementary Figure S1A–C). Upon exposure to high glucose, CD248 expression increased time-dependently in these cells (Figure 1d–f; Supplementary Figure S1A–C). To test whether CD248 induction is cell type–specific, we analyzed expression of CD248 transcript in human renal mesangial cells, h.GECs, and h.PODs stimulated with normal (5 mM) or high (25 mM) glucose or mannitol (25 mM). High glucose–induced CD248 expression was restricted to mesangial cells (Supplementary Figure S1D), corroborating earlier results.25Wilson P.C. Wu H. Kirita Y. et al.The single-cell transcriptomic landscape of early human diabetic nephropathy.Proc Natl Acad Sci U S A. 2019; 116: 19619-19625Crossref PubMed Scopus (266) Google Scholar Similar results were obtained in murine MES13 mesangial cells, as compared to murine glomerular endothelial cells (GECs), murine podocytes (PODs), and murine proximal tubular cells (BUMPT; Supplementary Figure S1E). Our data indicate that glucose-associated glomerular induction of CD248 is restricted to mesangial cells. These results demonstrate that the expression of CD248 and genes associated with UPR signaling is coordinately regulated in glucose-stressed mesangial cells in vitro. Considering the expression of CD248 in mesangial cells,25Wilson P.C. Wu H. Kirita Y. et al.The single-cell transcriptomic landscape of early human diabetic nephropathy.Proc Natl Acad Sci U S A. 2019; 116: 19619-19625Crossref PubMed Scopus (266) Google Scholar,26Smith S.W. Eardley K.S. Croft A.P. et al.CD248+ stromal cells are associated with progressive chronic kidney disease.Kid Int. 2011; 80: 199-207Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar we investigated CD248 expression in renal glomeruli isolated from STZ-induced diabetic mice and db/db mice (models of type 1 and type 2 diabetes mellitus, respectively; Supplementary Figure S2A and B). Glomerular CD248 transcript and protein expression was increased in mice in both diabetes models compared to that in nondiabetic control mice (Figure 2a–f ). Upregulation of CD248 in mesangial cells was confirmed in both murine diabetes models by costaining of CD248 with the mesangial cell markers α–smooth muscle actin and platelet-derived growth factor receptor beta (Figure 2g–i; Supplementary Figure S2C–E). To evaluate the relevance of this finding for DKD in humans, we analyzed human kidney samples from healthy controls and diabetic patients with versus without DKD. Based on costaining analysis, CD248 expression was markedly induced in the mesangial cells of diabetic patients with DKD, compared to that in diabetic patients without DKD and healthy controls (Figure 2j and k; Supplementary Figure S2F and G; Supplementary Table S2). Time course analysis revealed that increased glomerular expression of CD248 preceded the onset of albuminuria (Figure 3a–d ) as well as mesangial matrix expansion (Figure 3e–h) in both diabetes models (STZ and db/db). Although glomerular CD248 induction in murine type 1 diabetes and type 2 diabetes occurred at 8 weeks post–STZ administration and at 4 weeks of age, respectively, albuminuria onset was detectable at 12 weeks post–STZ administration in the type 1 diabetes model, and at 8 weeks of age in the type 2 diabetes model (Figure 3a–d). Similarly, the increase in mesangial matrix expansion, as determined by the fractional mesangial area, followed glomerular CD248 elevation (Figure 3e–h), suggesting that CD248 induction precedes DKD-associated glomerular dysfunction. To evaluate the functional relevance of CD248 in DKD, we induced persistent hyperglycemia in wild-type (CD248+/+) and CD248 global knockout (CD248–/–) mice by STZ administration. Despite the comparable blood glucose levels between wild-type and CD248–/– mice (Supplementary Figure S3A), albuminuria was markedly reduced in diabetic CD248–/– mice compared to diabetic wild-type mice at 10, 16, and 25 weeks post–STZ administration (Figure 4a ; Supplementary Figure S3B and C). Similarly, blood urea nitrogen and plasma creatinine levels were lower in diabetic CD248–/– mice (Figure 4b; Supplementary Figure S3D). Moreover, DKD-associated glomerular damage, as reflected by the increased fractional mesangial area, loss of podocytes, increased glomerular basement membrane width, decreased number of podocyte foot processes, and increased tight slit pore density, was markedly reduced in diabetic CD248–/– mice (Figure 4c–h). Although we observed only partial protection against albuminuria and reduced glomerular filtration, the morphologic indices in CD248–/– mice were undistinguishable from those in nondiabetic control mice. These results indicate that CD248 promotes glomerular dysfunction in DKD through an undefined mechanism. Given that CD248 is expressed by mesangial cells in renal glomeruli and that high glucose induces extracellular matrix gene expression and profibrotic transforming growth factor (TGF)β1 signaling in mesangial cells, we analyzed corresponding endpoints in CD248-deficient and control mesangial cells. We determined expression of the ECM genes fibronectin (Fn), collagen 1α2 (Col 1α2), Laminin, and Tgfβ1, TGFβ1-secretion, and phosphorylation of SMAD2 (pSMAD2). High glucose (but not the osmotic control mannitol) readily induced ECM genes and Tgfβ1 mRNA expression, TGFβ1 secretion, as well as pSMAD2 levels in primary mesangial cells obtained from CD248+/+, but not in those isolated from CD248–/– mice (Supplementary Figure S4A–F). Corresponding changes were observed in CD248 control (scr) and CD248 knockdown (shCD248) mesangial cells (Supplementary Figure S4G–K). These results reflect typical phenotypic changes in glucose-stressed mesangial cells, which are regulated in a CD248-dependent fashion. Although CD248 was the most strongly induced gene in gene expression analysis, pathways associated with the ER stress response were the most strongly upregulated pathways in glucose-stressed mesangial cells. This finding raises the question of whether CD248 is related to ER signaling. Induction of glomerular CD248 expression in both STZ-induced and db/db diabetic mice was indeed associated with loss of IRE1α phosphorylation, reduced XBP1 splicing, and increased nuclear levels of cleaved ATF6 and CHOP (Supplement